CN107998404B

CN107998404B - Folic acid targeting vector loaded with anticancer drug and preparation method and application thereof

Info

Publication number: CN107998404B
Application number: CN201711314545.XA
Authority: CN
Inventors: 雷群芳; 方文军; 郭永胜; 施浙琪; 陈雪瑞; 张莉
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2017-12-09
Filing date: 2017-12-09
Publication date: 2021-05-04
Anticipated expiration: 2037-12-09
Also published as: CN107998404A

Abstract

The invention discloses a folic acid targeting carrier loaded with anticancer drugs, and a preparation method and application thereof. Firstly, zinc nitrate and 2-methylimidazole are taken as carrier materials, an anticancer drug is encapsulated in the carrier by an original taste embedding method, and the anticancer drug-zeolite imidazole framework carrier is synthesized; and then the folic acid is connected through a coordination bond formed by zinc ions in the anticancer drug-zeolite imidazole skeleton carrier and folic acid-polyethylene glycol, and the folic acid targeting carrier loaded with the anticancer drug is successfully prepared. The targeted drug carrier has good biocompatibility, simple synthesis method and targeting property, and can reduce the toxic and side effects of the drug. Meanwhile, the drug loading of chloroquine phosphate in the carrier is up to 18%, and the utilization rate and the drug effect of the drug are improved.

Description

Folic acid targeting vector loaded with anticancer drug and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a folic acid targeting carrier loaded with an anticancer drug, and a preparation method and application thereof.

Background

The increasing global risk of cancer has become the first killer threatening the health of human beings. Currently, the most used treatment methods in the clinic are surgery, chemotherapy and also radiotherapy. Chemotherapy causes a range of acute and chronic organ toxicities. Since many chemotherapeutic drugs and their metabolites are excreted through renal tubular epithelial cells, the kidneys are easily damaged by chemotherapeutic drugs. Thus, the use of chemotherapeutic agents is sometimes limited by their nephrotoxic side effects. Chemotherapy-related kidney damage often results in inadequate cancer treatment because renal dysfunction requires the clinician to reduce the dose of chemotherapy to avoid further kidney damage. Kidney damage can also cause other adverse complications such as water and nitrogenous waste retention, electrolyte disturbance, decreased immunity, and the like.

The development of effective drug carriers, the reduction of the inherent adverse reactions of chemotherapeutic drugs and the improvement of the treatment effect become one of the key problems for treating cancers. Currently, drug nanocarriers, including polymersomes, micelles, polymeric nanoparticles, inorganic nanoparticles and hybrid porous solids, have been developed to improve drug accumulation in tumor regions for enhanced permeability and retention effects. Among them, nano Metal Organic Frameworks (MOFs) are attracting attention as drug nanocarriers with high drug loading, high pore volume, large surface area and adjustable pore size within the framework. Among the MOFs, the zeolitic imidazolate framework ZIF-8 is particularly promising as a drug carrier, since ZIF-8 is a nontoxic and biocompatible MOF built from zinc ions and 2-methylimidazolium salt, which is stable under physiological conditions and decomposes at tumor sites with low pH.

Chloroquine has shown a favourable effect as a novel antineoplastic drug in some clinical trials already in progress or ongoing. Although the exact mechanism remains to be determined, the anti-cancer effect of chloroquine may be due to its inhibitory effect on autophagy. Autophagy is one of the physiological processes affected by chloroquine. Chloroquine inhibits lysosomal activity, which in turn prevents the latter step of autophagy, i.e., degradation of the autophagosomes, resulting in an inability to provide energy via the autophagy pathway. Since autophagy is considered to be a pathway for cell survival in cancer, chloroquine has been used in combination with various chemotherapeutic agents and radiation therapy and has been shown to enhance killing of tumor cells, chloroquine phosphate (CQ), one of chloroquine.

Folate has been extensively studied as a targeting ligand for drug delivery, which can be selectively recognized by folate receptors overexpressed on the surface of cancer cells and stimulate receptor-mediated endocytosis, thereby increasing drug uptake by tumors while reducing systemic toxicity. Polyethylene glycol (PEG) is considered the best biocompatible material, prolonging circulation time. Therefore, folate-polyethylene glycol is considered to be one of the promising stabilizers to improve drug delivery systems to achieve satisfactory tumor-targeted therapy.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a folic acid targeting carrier loaded with an anticancer drug, and a preparation method and application thereof.

The folic acid targeting carrier loaded with the anticancer drugs is characterized in that zinc nitrate, 2-methylimidazole and the anticancer drugs are synthesized by a chemical method to obtain the anticancer drug-zeolite imidazole framework carrier; the anticancer drug-zeolite imidazole skeleton carrier is connected with folic acid through a coordination bond formed by zinc ions and folic acid-polyethylene glycol to obtain the folic acid targeting carrier loaded with the anticancer drug.

The folic acid targeting carrier loaded with the anticancer drug is characterized in that the anticancer drug is chloroquine phosphate.

The folic acid targeting carrier loaded with the anticancer drugs is characterized in that the chemical method is an in-situ embedding method.

The folic acid targeting carrier loaded with the anticancer drugs is characterized in that the anticancer drugs are encapsulated in a zeolite imidazole framework synthesized by zinc nitrate and 2-methylimidazole by adopting an in-situ embedding method.

The preparation method of the folic acid targeting carrier loaded with the anticancer drugs is characterized by comprising the following steps:

1) firstly, forming a coordination compound by utilizing zinc ions of zinc nitrate and an anticancer drug, then adding 2-methylimidazole and the zinc ions of the coordination compound into the coordination compound to form ZIF-8, and embedding the anticancer drug into the ZIF-8 by an in-situ embedding method to obtain an anticancer drug-zeolite imidazole framework carrier;

2) forming a coordination bond between zinc ions on the anticancer drug-zeolite imidazole framework carrier obtained in the step 1) and folic acid-polyethylene glycol, and connecting folic acid to the surface of the zeolite imidazole framework carrier to obtain the folic acid targeting carrier carrying the anticancer drug chloroquine phosphate.

The preparation method of the folic acid targeting carrier loaded with the anticancer drugs is characterized by comprising the following specific steps:

1) dissolving an anticancer drug in deionized water, mixing with zinc nitrate in water, stirring for 1-5min at room temperature to obtain a coordination compound system, adding anhydrous methanol and 2-methylimidazole into the coordination compound system, continuously stirring for 15-20min at room temperature to obtain a mixed system, transferring the mixed system into a centrifugal machine, centrifuging for 10-20min at 10000-12000rmp/min, washing the obtained solid with anhydrous methanol and deionized water respectively to remove unreacted reagents, and drying in vacuum to obtain the anticancer drug-ZIF-8 carrier, wherein preferably, the times of washing with anhydrous methanol and deionized water are both 3 times;

2) ultrasonically oscillating folic acid-polyethylene glycol and deionized water uniformly, adding the anticancer drug-ZIF-8 carrier obtained in the step 1), ultrasonically oscillating uniformly, stirring at room temperature for 35-50h, centrifugally washing to remove unreacted folic acid-polyethylene glycol, and vacuum drying to obtain the folic acid targeted carrier loaded with the anticancer drug.

The preparation method of the folic acid targeting carrier loaded with the anticancer drugs is characterized in that the mass ratio of the anticancer drugs, the zinc nitrate and the deionized water in the step 1) is 35-55: 1-2: 4-6, preferably 55:1:5 each; the mass volume ratio of the 2-methylimidazole to the anhydrous methanol is 2-5 g: 10ml, preferably 4 g: 8ml of the solution; mixing the anticancer drug and zinc nitrate, stirring at room temperature for 2min, adding anhydrous methanol and 2-methylimidazole into the coordination compound system, and stirring at room temperature for 16 min; the centrifugal speed is 11000rmp/min, and the centrifugal time is 10 min.

The preparation method of the folic acid targeting carrier loaded with the anticancer drugs is characterized in that the mass-volume ratio of the folic acid-polyethylene glycol to the deionized water in the step 2) is 8-10 g: 5ml, preferably 10 g: 1 ml.

The preparation method of the anticancer drug loaded folic acid targeting carrier is characterized in that the mass ratio of the folic acid-polyethylene glycol to the anticancer drug-ZIF-8 carrier in the step 2) is 2:2-3, preferably 1: 1.

The folic acid targeting carrier loaded with the anticancer drug is applied to the preparation of the anticancer drug.

By adopting the technology, compared with the prior art, the invention has the following beneficial effects:

1) the invention encapsulates the anticancer drug (chloroquine phosphate) in ZIF-8 by chemical reaction, and simultaneously, a CQ-ZIF-8 carrier is connected with folic acid-polyethylene glycol to construct a target drug carrier (FA-PEG/CQ-ZIF-8) and the material characterization is carried out, and the adopted characterization method comprises the following steps: fourier infrared spectroscopy (FTIR), X-ray diffraction (XRD), a scanning electron microscope and the like prove that the carrier is successfully constructed, and meanwhile, the toxicity of chloroquine phosphate and the FA-PEG/CQ-ZIF-8 carrier to cancer cells and normal cells is also researched, the survival rate of the carrier in the cancer cells and the normal cells is detected by utilizing an MTT contrast method, and the ZIF-8 in the carrier drug is proved to have a protective effect on the anti-cancer drug, so that the anti-cancer drug can reach a focus, and the chloroquine phosphate is released to treat the cancer, so that the toxicity to the cancer cells can be enhanced in a targeted manner, and the toxicity to the normal cells can be reduced; the solid foundation is laid for improving the drug loading rate of chloroquine phosphate, establishing a targeted delivery system of an anticancer drug and reducing the toxicity of organs, the problem of low utilization rate of the anticancer drug chloroquine phosphate is solved, the toxic and side effects of treating cancer are reduced, the drug treatment has targeting property, and a new way is developed for the treatment of cancer;

2) the zeolite imidazole framework ZIF-8 used in the invention is applied to a drug carrier system for the first time, the diameter of the ZIF-8 is 10-500 nanometers, and the ZIF-8 can enter cells through endocytosis of cell membranes, and has no cytotoxicity because of stable structure and capability of being discharged through human metabolism; ZIF-8 has huge specific surface area and pore volume, thereby having larger drug-loading capacity, and the special pore channel structure ensures that the ZIF-8 has the function of drug slow release;

3) the results of anticancer cell tests prove that the folic acid targeting vector loaded with the anticancer drug chloroquine phosphate is successfully prepared, the anticancer drug chloroquine phosphate can be carried to a focus in a targeted manner, the drug cytotoxicity can be reduced, the effect of the anticancer drug chloroquine phosphate on cancer cells is successfully reserved, and the folic acid targeting vector has a good inhibition effect and a good curative effect on the cancer cells.

Drawings

FIG. 1 is a standard graph of chloroquine phosphate;

FIG. 2A is an IR spectrum of ZIF-8;

FIG. 2B is an IR spectrum of FA-PEG;

FIG. 2C is an infrared spectrum of CQ;

FIG. 2D is an IR spectrum of FA-PEG/CQ-ZIF-8;

FIG. 3A is a transmission electron micrograph of a zeolitic imidazolate framework (ZIF-8);

FIG. 3B is a transmission electron microscope image of chloroquine phosphate loaded zeolitic imidazolate framework (CQ-ZIF-8);

FIG. 3C is a transmission electron micrograph of folate-polyethylene glycol/chloroquine phosphate-zeolitic imidazolate framework vector (FA-PEG/CQ-ZIF-8);

FIG. 4 is a UV fluorescence detection of CQ-ZIF-8;

FIG. 5A is a graph showing the results of cytotoxicity of folate-polyethylene glycol;

FIG. 5B is a graph showing the results of cytotoxicity of ZIF-8;

FIG. 5C is a graph showing the results of cytotoxicity of chloroquine phosphate;

FIG. 5D is a graph showing cytotoxicity results of CQ-ZIF-8 and FA-PEG/CQ-ZIF-8.

Detailed Description

EXAMPLE 1 preparation of folate targeting vector loaded with anticancer drug chloroquine phosphate

1. Preparation of chloroquine phosphate-zeolimidazole skeleton carrier (CQ-ZIF-8)

An anticancer drug chloroquine phosphate is encapsulated in a zeolite imidazole framework by an in-situ embedding method, and the chloroquine phosphate-zeolite imidazole framework carrier (CQ-ZIF-8) is synthesized, and the specific reaction is as follows:

(1) dissolving 50mg chloroquine phosphate in 5ml deionized water, mixing with zinc nitrate, stirring at room temperature for 1min, and forming a coordination compound by using zinc ions of the zinc nitrate and the anticancer drug;

(2) adding 8ml of anhydrous methanol and 4g of 2-methylimidazole, and continuously stirring for 15min at room temperature to form ZIF-8 by using 2-methylimidazole and zinc ions of the coordination compound;

(3) centrifuging at 10000rmp/min for 10min, washing with anhydrous methanol and deionized water for three times to remove unreacted reagent, and vacuum drying to obtain CQ-ZIF-8 carrier;

2. preparation of Folic acid-polyethylene glycol/chloroquine phosphate-Zeolite imidazole framework vector (FA-PEG/CQ-ZIF-8)

Zinc ions in a zeolite imidazole framework and folic acid-polyethylene glycol form a coordination bond to connect folic acid, and the specific reaction is as follows:

(1) carrying out ultrasonic oscillation on 50mg of folic acid-polyethylene glycol and 5ml of deionized water uniformly;

(2) adding 50mg CQ-ZIF-8 carrier, uniformly oscillating by ultrasonic wave, and stirring at room temperature for 35 h;

(3) centrifuging, washing to remove unreacted folic acid-polyethylene glycol, and vacuum drying to obtain FA-PEG/CQ-ZIF-8 carrier.

Example 2 characterization of the vectors FA-PEG/CQ-ZIF-8

1. The standard curve of chloroquine phosphate is shown in figure 1

Measuring absorbance values of chloroquine phosphate solution with different concentrations at 257nm by ultraviolet spectrophotometry to obtain the standard curve shown in FIG. 1, R shown in FIG. 1²0.9988 reaches above the 0.99 level, so this criterion is applicable. However, the standard curve is used in the range of 10-40. mu.g/ml.

2. Infrared spectroscopy detection

(1) Drying FA-PEG, CQ, ZIF-8, FA-PEG/CQ-ZIF-8, then placing into a mortar, adding a certain amount (much) of KBr, grinding uniformly to make the mixture to have a particle size less than 2 μm so as to avoid the influence of scattered light, then placing into a dryer for drying, pressing the mixture into a transparent sheet on an oil press under the pressure of about 40MPa, and measuring on the machine;

(2) the infrared spectrum (FTIR) is shown in figure 2A, figure 2B, figure 2C and figure 2D, wherein figure 2A is zeolite imidazole skeleton (ZIF-8), figure 2B is folic acid-polyethylene glycol (FA-PEG), figure 2C is chloroquine phosphate (CQ), figure 2D is folic acid-polyethylene glycol/chloroquine phosphate-zeolite imidazole skeleton carrier (FA-PEG/CQ-ZIF-8), and figure 2A has an absorption peak at 1570 to represent-C = C-; a stronger absorption peak at 1420 indicates-N = C-, these groups should be present in ZIF-8; FIG. 2B shows absorption peaks at 3570, 1650, and 1600 indicating-COOH; FIG. 2C shows a characteristic absorption peak 1090 representing the absorption peak of chlorobenzene; in FIG. 2D after synthesis, there is an absorption peak at 3570 representing-H on folate carboxylate, which is grafted with folate-polyethylene glycol; an absorption peak at 1420 represents-N = C-with ZIF-8; the characteristic absorption peak of fig. 2C is not seen because the drug is encapsulated inside the carrier.

3. The transmission electron microscope images of ZIF-8, CQ-ZIF-8 and FA-PEG/CQ-ZIF-8 are shown in figures 3A, 3B and 3C, wherein figure 3A is a zeolimidazole skeleton (ZIF-8), figure 3B is a chloroquine phosphate loaded zeolimidazole skeleton (CQ-ZIF-8), and figure 3C is a folic acid-polyethylene glycol/chloroquine phosphate-zeolimidazole skeleton carrier (FA-PEG/CQ-ZIF-8).

The transmission electron microscope image shows that the zeolite imidazole skeleton is octahedron and the size is in the nanometer level; as can be seen from a comparison of FIGS. 3A and 3B, when ZIF-8 was synthesized separately from drug-loaded ZIF-8, the octahedral crystal form was more pronounced, and the particle size was reduced and no longer sticky. Comparing the graph B with the graph C, the CQ-ZIF-8 crystal forms before and after the inoculation of the folic acid are basically unchanged and are relatively stable.

EXAMPLE 3 ZIF-8 loaded CQ Loading Rate in FA-PEG/CQ-ZIF-8 vectors

1. Measurement of CQ Loading Rate of ZIF-8 load

Detecting by an ultraviolet spectrophotometer: the characteristic absorption peak of chloroquine phosphate is 254nm according to the literature

(1) The specific steps for drawing the standard curve of chloroquine phosphate are as follows:

preparation of curcumin stock solution

Accurately weighing 1mg of chloroquine phosphate, placing the chloroquine phosphate in a beaker, adding a proper amount of deionized water to dissolve the chloroquine phosphate, transferring the chloroquine phosphate into a 10mL volumetric flask, and then using the deionized water to perform constant volume to a scale for later use as a chloroquine phosphate stock solution;

② 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0mL of the stock solution are respectively weighed and placed in 7 10mL volumetric flasks, the volume is determined to the scale with deionized water, and the mixture is shaken up. Taking deionized water as a blank control, measuring the absorbance at 257nm, and drawing a calibration curve by taking the average value (A) of the three absorbances of each concentration as the ordinate and the concentration (c) as the abscissa;

(2) measurement of CQ Loading Rate of ZIF-8 load

Adding 0.9% hydrochloric acid solution into the prepared CQ-ZIF-8, and dissolving ZIF-8 so as to dissolve chloroquine phosphate in the solution;

measuring the light absorption value of the CQ-ZIF-8 hydrochloric acid solution at 257nm, and calculating the loading rate of chloroquine phosphate in the solution according to a standard curve;

the ultraviolet fluorescence detection of CQ-ZIF-8 is shown in figure 4, and the drug loading rate can be up to 18% according to the 257nm absorption peak.

Example 4 cytotoxicity assays

Carrying out ultraviolet lamp sterilization treatment on CQ, CQ-ZIF-8 and FA-PEG/CQ-ZIF-8, respectively dissolving the sterilized materials in ultrapure water, and placing the ultrapure water in a 50 mL volumetric flask to prepare a solution with the concentration of 200 mug/mL. Diluting CQ, CQ-ZIF-8, FA-PEG/CQ-ZIF-8 solution to desired concentration (0.1, 0.5, 1, 2, 5, 10, 15, 20 μ g/ml) with DMEM culture medium by adopting concentration gradient stepwise dilution method,

(1) taking HeLa cells grown in logarithmic phase, digesting the monolayer cells with 0.25% trypsin to prepare a single cell suspension (medium: DMEM +10% FCS), adding 200. mu.L of the cell suspension to the cell test wells of a 96-well plate, and multiplying 1X 10⁴Cells/well, placing cells in CO₂Incubator (37 ℃, 5% CO)₂) Medium culture is carried out for 16-18h to achieve complete adherence;

(2) the culture medium in the 96-well plate was aspirated by a pipette, 200 μ L of the culture medium containing CQ or CQ-ZIF-8 or FA-PEG/CQ-ZIF-8 at different concentrations was added to the test wells, and the blank control group was added with the corresponding culture medium and sterile water, respectively. All control and test groups were run in parallel 5 times. Continuing to culture the cells in CO₂Incubator (37 ℃, 5% CO)₂) Neutralizing for 4 h;

(3) discarding the culture medium, adding 100 mu L of 5 mg/mL MTT solution into each well, and continuing to add CO₂Incubator (37 ℃, 5% CO)₂) Culturing for 4 h;

(4) the plates were turned to discard the supernatant and 100. mu.L DMSO was added to each well. Oscillating for 5min on an oscillator, and measuring the absorbance OD value of the solution at the wavelength of 570nm by using an enzyme-labeling instrument;

the survival rate (V) of the cells in different surfactant solutions was calculated according to the following formula:

V=（A-A₀）/（A_C-A₀）

wherein:

v is the survival (%) of the cells;

a is the OD value of the cells cultured by 3-MA @ ZIF-8 solution;

A₀the OD value of the cell is 0 after CQ or CQ-ZIF-8 or FA-PEG/CQ-ZIF-8 is replaced by sterilized water;

ac is the OD value of the cells without CQ or CQ-ZIF-8 or FA-PEG/CQ-ZIF-8, and the cell growth rate is 100%.

(5) The cytotoxicity test result graphs are shown in fig. 5A, 5B, 5C and 5D, in which fig. 5A is a graph showing the cytotoxicity result of folate-polyethylene glycol, fig. 5B is a graph showing the cytotoxicity result of ZIF-8, fig. 5C is a graph showing the cytotoxicity result of chloroquine phosphate, fig. 5D is a graph showing the cytotoxicity result of CQ-ZIF-8, and B is a graph showing the cytotoxicity result of FA-PEG/CQ-ZIF-8.

According to FIG. 5A, it can be obtained that FA-PEG has very little cytotoxicity and the cell survival rate is at the reagent concentration (IC) corresponding to 50%₅₀Value) is 10 g/ml; FIG. 5A compares FIG. 5B and FIG. 5C, FA-PEG cytotoxicity was negligible, and IC of CQ₅₀IC with significantly higher value than ZIF-8₅₀The value is large, and the visible drug has higher toxicity than the carrier; from a comparison of a and b in FIG. 5D, the IC of b can be observed₅₀The value was 18. mu.g/ml less than that of FIG. a, indicating that the folate-conjugated vehicle provides targeting of the drug.

Claims

1. The preparation method of the folic acid targeting carrier loaded with the anticancer drugs is characterized by comprising the following steps:

1) preparation of chloroquine phosphate-zeolite imidazole framework carrier CQ-ZIF-8 the chloroquine phosphate as an anticancer drug is encapsulated in a zeolite imidazole framework by an in-situ embedding method to synthesize the chloroquine phosphate-zeolite imidazole framework carrier (CQ-ZIF-8), and the specific reaction is as follows:

1.1) dissolving 50mg of chloroquine phosphate in 5ml of deionized water, mixing with zinc nitrate, stirring for 1min at room temperature, and forming a coordination compound by utilizing zinc ions of the zinc nitrate and the chloroquine phosphate;

1.2) adding 8ml of anhydrous methanol and 4g of 2-methylimidazole, and continuously stirring for 15min at room temperature to enable the 2-methylimidazole and zinc ions of a coordination compound to form ZIF-8;

1.3) centrifuging for 10min at the rotating speed of 10000rmp/min, washing for three times by using anhydrous methanol and deionized water respectively to remove unreacted reagents, and drying in vacuum to obtain a CQ-ZIF-8 carrier;

2) the zinc ions in the zeolite imidazole framework prepared by the folic acid-polyethylene glycol/chloroquine phosphate-zeolite imidazole framework carrier FA-PEG/CQ-ZIF-8 and the folic acid-polyethylene glycol form coordinate bonds to connect folic acid, and the specific reaction is as follows:

2.1) carrying out ultrasonic oscillation on 50mg of folic acid-polyethylene glycol and 5ml of deionized water uniformly;

2.2) adding 50mg CQ-ZIF-8 carrier, uniformly oscillating by ultrasonic wave, and stirring for 35h at room temperature;

2.3) centrifuging, washing and removing unreacted folic acid-polyethylene glycol, and drying in vacuum to obtain the FA-PEG/CQ-ZIF-8 carrier.